INTRODUCTION:

Chemically Levocetirizine dihydrochloride is [2-[4-(R)-(4-Chlorophenyl) phenyl methyl]-1-Piperazinyl] ethoxy]-acetic acid dihydrochloride. Is a selective potent H1-antihistamine compound indicated for the treatment of allergic rhinitis, mild sedative, mucosal decongestant and chronic idiopathic urticaria and is used to reduce itchy skin rash and hives. Analysis is an important component in the formulation development of any drug molecule. A suitable and validated method has to be available for the analysis of drug(s) in the bulk, in drug delivery systems, from release dissolution studies and in biological samples. If a suitable method, for specific need, is not available then it becomes essential to develop a simple, sensitive, accurate, precise, reproducible method for the estimation of drug samples the estimation of levocetirizine dihydrochloride by mercurimetric method, high performance liquid chromatography [HPLC], and high performance thin layer chromatography [HPTLC]^3-6 and spectroflurimetric method is reported in literature.

Although simultaneous UV estimation of levocetirizine dihydrochloride and Pseudoephedrine hydrochloride, Mortia M.R, Berton D¹., Boldin R. has been reported but single estimation of this drug has not been reported in bulk and in pharmaceutical formulation.

Thus the present study was undertaken to develop and validate a simple, sensitive, accurate, precise, and reproducible U.V method for levocetirizine dihydrochloride.

Figure No.1 - Chemical structure of levocetirizine dihydrochloride².

MATERIALS AND METHOD:

Instrument and materials:

Instrument used was UV/Visible Spectrophotometer and schimadzu pharmaspec UV 1700 analytical balance. Levocetirizine dihydrochloride pure drug was obtain from Reddy’s Labs LTD. Hyderabad as gift sample with 99.99% w/w assay value and was used without further purification. All chemicals and reagents used were of analytical grade.

Preparation of standard stock solution:

Standard drug solution of levocetirizine dihydrochloride was prepared by dissolving 25mg levocetirizine dihydrochloride in 25ml methanol and transferred it to 25ml volumetric flask and volume was made up to mark with methanol to obtain stock solution of 1000μg/ml concentration. From that take 1 ml solution made up to 10 ml with methanol to obtain 100 µg/ml concentration. For obtaining clear solution, solution was ultrasonicated.

Preparation of calibration curve:

Calibration curve was prepared in methanol at λ max 229 nm using UV/Visible Spectrophotometer For this stock solution of 100 μg /ml was prepared. Serial dilution of 2, 4, 6, 8, 10μg/ml were prepared and absorbance was taken at λ max 229 nm. Averages of such 5 sets of values were taken for standard calibration curve, and solutions were scanned in the range of 200-400 nm against blank. The calibration curve was plotted. The optical characteristics are summarized in Table no.1

Table No.1 - Calibration Curve

S. No	Concentration ( μg/ml)	Absorbance
1	2	0.048
2	4	0.11
3	6	0.16
4	8	0.19
5	10	0.27

Table No .2 - Validation parameters

S. No.	Parameter	Result
1	Absorption maxima(nm)	229
2	Linearity Range (μg/ml)	2-10
3	Standard Regression Equation	y = 0.026x + 0.016
4	Correlation Coefficient (r²)	0.999
5	Molar absorptivity	51578.13
6	Accuracy (% recovery )	98.8
7	Precision	99.50% (Intra-day precision) and 99.60%(Inter-day precision)
8	Specificity	A 30μ g/ml solution of candidate drug in methanol at UV detection l of 229 nm will show an absorbance value of 0.515
9	LOD (μg/ml)	2.7
10	LOQ (μg/ml)	8.2

Preparation of sample solution:

Ten tablets were weighed and powdered. The amount of tablet powder equivalent to 25mg of levocetirizine dihydrochloride was weighed accurately and transferred to 100ml volumetric flask containing methanol and the volume was made up to the mark .The solution was filtered through whatmann filter paper no.4. Adequate quantity of solution was diluted to get a concentration 30μg/ml of levocetirizine dihydrochloride. The absorbance was measured against blank. The drug content of the preparation was calculated using standard calibration curve. Amount of drug estimated by this method is given in Table no.3.

RESULT AND DISCUSSION:

Accuracy (Recovery Test):

Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts to tablet. The recovery was performed at three levels, 80, 100 and 120% of levocetirizine dihydrochloride standard concentration. The recovery samples were prepared in afore mentioned procedure. Three samples were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The recovery values for levocetirizine dihydrochloride ranged from 99.97 Table no.3.

Figure No.2 - Determination of λ max of levocetirizine dihydrochloride by UV scanning.

Precision:

Assay of method precision (intra-day precision) was evaluated by carrying out three independent assays of test samples of levocetirizine dihydrochloride. The intermediate precision (inter-day precision) of the method was also evaluated using two different analysts, systems and different days in the same laboratory. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.0055, 99.5 and 0.0016, 99.60 respectively Table no.4.

Linearity:

The linearity of the response of the drug was verified at 2 to 30 μg/ml concentrations, but linearity was found to be between 2-10μg/ml concentrations. The calibration graphs were obtained by plotting the absorbance versus the concentration data and were treated by linear regression analysis Table no.2. The equation of the calibration curve for levocetirizine dihydrochloride obtained y = 0.026x + 0.016, the calibration curve was found to be linear in the afore mentioned concentrations. The correlation coefficient (r²) of determination was 0.99.

Figure No.3 - Determination of linearity of levocetirizine dihydrochloride.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ of Levocetirizine dihydrochloride were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines .The LOD and LOQ was found to be as in Table no.2.

Determination of Active Ingredients in Tablets:

The validated method was applied to the determination of Levocetirizine dihydrochloride in Tablets. Ten tablets were assayed and the results are shown in Table no. 3 indicating that the amount of drug in tablet samples met with requirements (99–102% of the label claim).

Table No. 4 - Determination of Precision

Sample number	Assay of Levocetirizine dihydrochloride as % of labeled amount
	Analyst-I (Intra-day precision)	Analyst-II (Inter-day precision)
1	99.42	99.70
2	99.63	99.23
3	99.58	99.57
4	99.10	99.88
5	100.12	99.98
6	99.20	99.25
Mean	99.50	99.60
RSD	0.54	0.43

CONCLUSIONS:

The developed method was found to be simple, sensitive, accurate, precise, reproducible, and can be used for routine quality control analysis of Levocetirizine dihydrochloride in bulk and pharmaceutical formulation

ACKNOWLEDGEMENT:

We are thankful to Reddy’s lab Ltd. Hyderabad for providing the gift sample of levocetirizine dihydrochloride. We would also like to thank Mr. Ishari Ganesh, Chancellor of Vels University, Chennai 117, for providing all the facilities to complete our work successfully.

REFERENCES:

1. Mortia M.R, Berton D., Boldin R., Journal of chromatography B, 2008; 862:132-139

2. Indian Pharmacopoeia, Vol. II, Government of India, , New Delhi, 2007, 1290,

3. Sweetman, S.C., Martindale, the Extra pharmacopoeia, 34thed.London: pharmaceutical Press, 2004, 435.3, and 1129. 2.

4. A.F.M. El Walily., Korany, M.A., Gindy, M.F., J.Pharmaceut.Biomed.Anal.17, 1998, 435–442.

5. Sevgi K., J.Pharmaceut.Biomed.Anal; 46 (2008) 295–302.

6. Sun Ok Choi. Seok Ho Lee., Hak Soo Kong., Eun Jung Kim., Hae-Young Park Choo, Journal of Chromatography B, 744, 2000, 201–206.

Received on 22.10.2009 Modified on 10.02.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 539-541

Ingredient	Tablet amount(µg/ml)	Level of addition (%)	Amount added (mg)	Drug found (mg/ml)	% Recovery	Average % recovery
Levocetirizine dihydrochloride*	30	80	0.5	4.89	97.8	98.8
	30	100	0.5	4.95	99.00
	30	120	0.5	4.98	99.6